CN111690750A - PCR reagent for detecting molecular expression of cell P53 signal path and application thereof - Google Patents

PCR reagent for detecting molecular expression of cell P53 signal path and application thereof Download PDF

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CN111690750A
CN111690750A CN202010666777.7A CN202010666777A CN111690750A CN 111690750 A CN111690750 A CN 111690750A CN 202010666777 A CN202010666777 A CN 202010666777A CN 111690750 A CN111690750 A CN 111690750A
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pcr reaction
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袁荣涛
仇静
唐永平
陈正岗
于江波
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Qingdao Municipal Hospital
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Abstract

The PCR reagent comprises PCR reaction primers for detecting related genes such as APAF1 gene, ATM gene, ATR gene, BAI1 gene, BAX gene and the like and reference genes. The invention concentrates the core signal molecules related to the tumor P53 signal channel on a flat plate, reacts the survival state of cells by carrying out a real-time fluorescent quantitative PCR reaction, compares the human oral squamous carcinoma cell SCC-25 with normal oral mucosa cells, discusses the regulation and control mode of the core molecules of the P53 signal channel in human tumors and normal cells, and provides the most direct evidence for researching the regulation and control of key proteins; the invention quickly and accurately finds the P53 signal channel core signal molecule in the tumor cell from the transcription level, and provides a powerful tool for mechanism discussion of new targeted drugs, development of targeted tumor inhibitors and the like.

Description

PCR reagent for detecting molecular expression of cell P53 signal path and application thereof
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a PCR reagent for detecting molecular expression of a cell P53 signal pathway and application thereof.
Background
The p53 gene is the gene that has been found to be most highly correlated with human tumors to date. p53 is mutated in most tumor cells, and more than 50% of all malignancies show mutations in this gene. The protein encoded by this gene is a transcription factor that controls the initiation of the cell cycle. Many signals about the health of cells are sent to the p53 protein, which determines whether a cell enters a division cycle. P53 is a tumor suppressor protein that regulates the expression of a wide variety of genes, including apoptosis, growth suppression, inhibition of cell cycle progression, differentiation and acceleration of DNA repair, genotoxicity and senescence following cellular stress. Like all other tumor suppressors, the p53 gene normally acts to slow or monitor cell division. In addition, p53 can act as a transcription factor to initiate the apoptotic pathway independently of its activity.
p53 is a star cancer suppressor gene that regulates cell cycle and prevents cell carcinogenesis, and is called "genomic guardian" by the scientific community, and is usually mutated and inactivated in p53 gene in more than 50% of cancer patients.
Studies accumulated over decades have found that mutations in the p53 signaling pathway have a greater impact on the incidence of different cancers than other signaling pathways, but the basis of these studies is based on somatic mutations. Scientists have recently taken advantage of the enormous genome data analysis to find that the role of genetic mutations in the p53 signaling pathway plays an important role in a range of cancers. As drugs that modulate p53 signaling are entering the clinical stage, the p 53-related signaling pathway molecules should be adequately considered when designing and testing cancer patient stratification strategies. Therefore, the research on P53 cell signal transduction pathway molecules in tumor cells confirms that the core factors playing a role in specific tumor tissues have important practical significance for treating tumors.
Disclosure of Invention
Aiming at determining the regulation and control of a P53 signal molecule and explaining the change of the core molecule in a tumor cell in the prior art, the invention provides a PCR reagent for detecting the molecular expression of a P53 signal channel of the cell and application thereof, wherein the invention concentrates the signal molecules related to the P53 signal channel on a flat plate, reacts the survival state of the cell by carrying out a real-time fluorescent quantitative PCR reaction, discusses the possible target molecules and target pathways causing the change of the P53 signal channel core molecule and provides the most direct evidence for researching the regulation of the P53 signal channel core molecule.
In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:
a PCR reagent for detecting the expression of cell P53 signal channel molecule, which comprises the following primers:
(1) the PCR reaction primers for detecting the APAF1 gene have the following primer sequences:
5'-CACACGGTTGGATCAGGAT-3'(SEQ ID NO:1)；
5'-GGAGACGGTCTTTAGCCTCT-3'(SEQ ID NO:2)；
(2) the PCR reaction primer for detecting the ATM gene has the following primer sequence:
5'-TGATCTTGTGCCTTGGCTAC-3'(SEQ ID NO:3)；
5'-ATGGTGTACGTTCCCCATGT-3'(SEQ ID NO:4)；
(3) the PCR reaction primers for detecting the ATR gene have the following primer sequences:
5'-CCCTTGAATACAGTGGCCTA-3'(SEQ ID NO:5)；
5'-CCTTGAAAGTACGGCAGTTC-3'(SEQ ID NO:6)；
(4) the PCR reaction primer for detecting the BAI1 gene has the following primer sequence:
5'-CAACCTGGTTCTCAGCATCC-3'(SEQ ID NO:7)；
5'-GACGGTCGTGTTCCTCTG-3'(SEQ ID NO:8)；
(5) the PCR reaction primer for detecting the BAX gene has the following primer sequence:
5'-CCGAGAGGTCTTTTTCCGAG-3'(SEQ ID NO:9)；
5'-CAGCCCATGATGGTTCTGAT-3'(SEQ ID NO:10)；
(6) the PCR reaction primers for detecting the BBC3 gene have the following primer sequences:
5'-ACCTCAACGCACAGTACGAG-3'(SEQ ID NO:11)；
5'-GGAGTCCCATGATGAGATTGT-3'(SEQ ID NO:12)；
(7) the PCR reaction primers for detecting the BCL2 gene have the following primer sequences:
5'-GTGGGGTCATGTGTGTGG-3'(SEQ ID NO:13)；
5'-GGTTCAGGTACTCAGTCATCC-3'(SEQ ID NO:14)；
(8) the PCR reaction primers for detecting the BCL2A1 gene have the following primer sequences:
5'-GTGCTACAAAATGTTGCGTTC-3'(SEQ ID NO:15)；
5'-GCAATTTGCTGTCGTAGAAGTT-3'(SEQ ID NO:16)；
(9) the PCR reaction primer for detecting BID gene has the following primer sequence:
5'-TGGACCGTAGCATCCCTCC-3'(SEQ ID NO:17)；
5'-TAGGTGCGTAGGTTCTGGT-3'(SEQ ID NO:18)；
(10) the PCR reaction primers for detecting the BIRC5 gene have the following primer sequences:
5'-GGACCACCGCATCTCTACAT-3'(SEQ ID NO:19)；
5'-AGTCTGGCTCGTTCTCAGTG-3'(SEQ ID NO:20)；
(11) the PCR reaction primers for detecting BRCA1 gene have the following primer sequences:
5'-TGTTACAAATCACCCCTCAAGG-3'(SEQ ID NO:21)；
5'-CCTGATACTTTTCTGGATGCC-3'(SEQ ID NO:22)；
(12) the PCR reaction primers for detecting BRCA2 gene have the following primer sequences:
5'-GCCTGAAAACCAGATGACTATC-3'(SEQ ID NO:23)；
5'-GGCCAGCAAACTTCCGTTTA-3'(SEQ ID NO:24)；
(13) the primer sequence of the PCR reaction primer for detecting the BTG2 gene is as follows:
5'-CTGTGGGTGGACCCCTAT-3'(SEQ ID NO:25)；
5'-GCCTCCTCGTACAAGACG-3'(SEQ ID NO:26)；
(14) the PCR reaction primers for detecting the CASP2 gene have the following primer sequences:
5'-GCTGTTGTTGAGCGAATTGT-3'(SEQ ID NO:27)；
5'-GCAAGTTGAGGAGTTCCACA-3'(SEQ ID NO:28)；
(15) the PCR reaction primers for detecting the CASP9 gene have the following primer sequences:
5'-TGTCTACGGCACAGATGGAT-3'(SEQ ID NO:29)；
5'-GGACTCGTCTTCAGGGGAA-3'(SEQ ID NO:30)；
(16) the PCR reaction primers for detecting the CCNB1 gene have the following primer sequences:
5'-ACTTTCGCCTGAGCCTATTTT-3'(SEQ ID NO:31)；
5'-TGGTCTGACTGCTTGCTCTT-3'(SEQ ID NO:32)；
(17) the PCR reaction primers for detecting the CCNE1 gene have the following primer sequences:
5'-CTCAACGTGCAAGCCTCG-3'(SEQ ID NO:33)；
5'-CTCAAGAAAGTGCTGATCCC-3'(SEQ ID NO:34)；
(18) the PCR reaction primers for detecting the CCNG1 gene have the following primer sequences:
5'-AGTCTGCACACGATAATGGC-3'(SEQ ID NO:35)；
5'-TGCTTGGGCTGTACCTTCA-3'(SEQ ID NO:36)；
(19) the PCR reaction primer for detecting the CCNH gene has the following primer sequence:
5'-GGCACTTGAACAGATACTGGA-3'(SEQ ID NO:37)；
5'-CAATATGGGATAGCGGGTCT-3'(SEQ ID NO:38)；
(20) PCR primers for detecting CDC25A gene have the following primer sequences:
5'-TCCTCTTTTTACACCCCAGTCA-3'(SEQ ID NO:39)；
5'-CGGTTGTCAAGGTTTGTAGTTC-3'(SEQ ID NO:40)；
(21) PCR primers for detecting CDC25C gene have the following primer sequences:
5'-TGACAATGGAAACTTGGTGGAC-3'(SEQ ID NO:41)；
5'-GAGCGATATAGGCCACTTCTG-3'(SEQ ID NO:42)；
(22) PCR primers for detecting CDK1 gene have the following primer sequences:
5'-GATGTGCTTATGCAGGATTCC-3'(SEQ ID NO:43)；
5'-ATGTACTGACCAGGAGGGATAG-3'(SEQ ID NO:44)；
(23) PCR primers for detecting CDK4 gene have the following primer sequences:
5'-TGGTGTTTGAGCATGTAGACC-3'(SEQ ID NO:45)；
5'-ATCCTTGATCGTTTCGGCTG-3'(SEQ ID NO:46)；
(24) the PCR reaction primers for detecting the CDKN1A gene have the following primer sequences:
5'-GATGGAACTTCGACTTTGTCA-3'(SEQ ID NO:47)；
5'-CACAAGGGTACAAGACAGTG-3'(SEQ ID NO:48)；
(25) the PCR reaction primers for detecting the CDKN2A gene have the following primer sequences:
5'-TGGAGCCTTCGGCTGACT-3'(SEQ ID NO:49)；
5'-TAACTATTCGGTGCGTTGGG-3'(SEQ ID NO:50)；
(26) the PCR reaction primers for detecting the CHEK1 gene have the following primer sequences:
5'-CTTACTGCAATGCTCGCTGG-3'(SEQ ID NO:51)；
5'-TGAGGGGTTTGTTGTACCATC-3'(SEQ ID NO:52)；
(27) the PCR reaction primers for detecting the CHEK2 gene have the following primer sequences:
5'-TATCTGCCTTAGTGGGTATCCA-3'(SEQ ID NO:53)；
5'-TGTCGTAAAACGTGCCTTTG-3'(SEQ ID NO:54)；
(28) the PCR reaction primer for detecting CRDD gene has the following primer sequence:
5'-ATCAGACCGGCAGATTAACC-3'(SEQ ID NO:55)；
5'-TTGGCCTTACAGCGGTAGAT-3'(SEQ ID NO:56)；
(29) the PCR reaction primers for detecting the DNMT1 gene have the following primer sequences:
5'-CTAGCCCCAGGATTACAAGG-3'(SEQ ID NO:57)；
5'-CTCATCCGATTTGGCTCTTTC-3'(SEQ ID NO:58)；
(30) the PCR reaction primers for detecting the E2F1 gene have the following primer sequences:
5'-ATCCCAGGAGGTCACTTCTG-3'(SEQ ID NO:59)；
5'-ACAACAGCGGTTCTTGCTC-3'(SEQ ID NO:60)；
(31) the PCR reaction primers for detecting the E2F3 gene have the following primer sequences:
5'-AAGCCCCTCCAGAAACAAGA-3'(SEQ ID NO:61)；
5'-CTTGGGTACTTGCCAAATGT-3'(SEQ ID NO:62)；
(32) the PCR reaction primers for detecting the EGFR gene have the following primer sequences:
5'-TGCCGCAAAGTGTGTAACG-3'(SEQ ID NO:63)；
5'-TGCCGCAAAGTGTGTAACG-3'(SEQ ID NO:64)；
(33) the PCR reaction primers for detecting the EGR1 gene have the following primer sequences:
5'-CCCCTCTGTCTACTATTAAGGC-3'(SEQ ID NO:65)；
5'-GGGACTGGTAGCTGGTATTG-3'(SEQ ID NO:66)；
(34) the PCR primers for detecting the EI24 gene have the following primer sequences:
5'-TCGGTAACAGCCCGAATTATC-3'(SEQ ID NO:67)；
5'-CACTGAAAATTGACGTGAGGAA-3'(SEQ ID NO:68)；
(35) the PCR reaction primer for detecting the ESR1 gene has the following primer sequence:
5'-AAAGGTGGGATACGAAAAGACC-3'(SEQ ID NO:69)；
5'-CTGTTCTTCTTAGAGCGTTTGA-3'(SEQ ID NO:70)；
(36) the primer sequence of the PCR primer for detecting the FADD gene is as follows:
5'-TGGCTGACCTGGTACAAGAG-3'(SEQ ID NO:71)；
5'-GTAGATGCGTCTGAGTTCCAT-3'(SEQ ID NO:72)；
(37) the primer sequence of the PCR reaction primer for detecting the FAS gene is as follows:
5'-GATTGTGTGATGAAGGACATGG-3'(SEQ ID NO:73)；
5'-GTTGCTGGTGAGTGTGCATT-3'(SEQ ID NO:74)；
(38) the primer sequence of the PCR reaction primer for detecting the FASLG gene is as follows:
5'-TTTAACAGGCAAGTCCAACTCA-3'(SEQ ID NO:75)；
5'-GCCACCCTTCTTATACTTCACT-3'(SEQ ID NO:76)；
(39) the primer sequence of the PCR reaction primer for detecting the FOXO3 gene is as follows:
5'-CGGCTGACTGATATGGCAG-3'(SEQ ID NO:77)；
5'-GTGATGTTATCCAGCAGGTC-3'(SEQ ID NO:78)；
(40) the PCR reaction primers for detecting the GADD45A gene have the following primer sequences:
5'-CCTGATCCAGGCGTTTTG-3'(SEQ ID NO:79)；
5'-ATCCATGTAGCGACTTTCCC-3'(SEQ ID NO:80)；
(41) the PCR reaction primer for detecting the GML gene has the following primer sequence:
5'-TCTTTGCCTTACTCCTAGCCA-3'(SEQ ID NO:81)；
5'-GCGCCTAATATGATACGGACA-3'(SEQ ID NO:82)；
(42) the primer sequence of the PCR reaction primer for detecting the HDAC1 gene is as follows:
5'-GCCCTCACAAAGCCAATG-3'(SEQ ID NO:83)；
5'-TGCTTGCTGTACTCCGACA-3'(SEQ ID NO:84)；
(43) the PCR reaction primers for detecting the HK2 gene have the following primer sequences:
5'-TGACCAGGAGATTGACATGGG-3'(SEQ ID NO:85)；
5'-AACCGCATCAGGACCTCA-3'(SEQ ID NO:86)；
(44) the PCR reaction primer for detecting IGF1R gene has the following primer sequence:
5'-TGCTGACCTCTGTTACCTCT-3'(SEQ ID NO:87)；
5'-GCTTATTCCCCACAATGTAGTT-3'(SEQ ID NO:88)；
(45) the primer sequence of the PCR reaction primer for detecting the IL6 gene is as follows:
5'-CTGAACCTTCCAAAGATGGC-3'(SEQ ID NO:89)；
5'-TCACCAGGCAAGTCTCCTCA-3'(SEQ ID NO:90)；
(46) the PCR reaction primer for detecting the JUN gene has the following primer sequence:
5'-ACAGGTGGCACAGCTTAAAC-3'(SEQ ID NO:91)；
5'-AACTGCTGCGTTAGCATGAG-3'(SEQ ID NO:92)；
(47) the PCR reaction primers for detecting the KAT2B gene have the following primer sequences:
5'-GGAAAACCTGTGGTTGAAGG-3'(SEQ ID NO:93)；
5'-AGTCTTCGTTGAGATGGTGC-3'(SEQ ID NO:94)；
(48) the primer sequence of the PCR reaction primer for detecting the KRAS gene is as follows:
5'-AGTACAGTGCAATGAGGGAC-3'(SEQ ID NO:95)；
5'-CTGAGCCTGTTTTGTGTCTAC-3'(SEQ ID NO:96)；
(49) the PCR reaction primers for detecting the MCL1 gene have the following primer sequences:
5'-TGCCTTTGTGGCTAAACACT-3'(SEQ ID NO:97)；
5'-GTCCCGTTTTGTCCTTACGA-3'(SEQ ID NO:98)；
(50) PCR primers for detecting MDM2 gene, the primer sequence is as follows:
5'-GCAGGGGAGAGTGATACAGA-3'(SEQ ID NO:99)；
5'-AAGCCAATTCTCACGAAGGG-3'(SEQ ID NO:100)；
(51) PCR primers for detecting MDM4 gene, the primer sequence is as follows:
5'-TTTGATCCCTGCAACTCAGTG-3'(SEQ ID NO:101)；
5'-TCTCGTGGTCTTTTCTCACAT-3'(SEQ ID NO:102)；
(52) the PCR reaction primers for detecting MLH1 gene have the following primer sequences:
5'-CAAACCCCTGTCCAGTCAG-3'(SEQ ID NO:103)；
5'-TGGGAGTTCAAGCATCTCCT-3'(SEQ ID NO:104)；
(53) the PCR reaction primers for detecting the MSH2 gene have the following primer sequences:
5'-GTCAGAGCCCTTAACCTTTTTC-3'(SEQ ID NO:105)；
5'-AGAGGCTGCTTAATCCACTG-3'(SEQ ID NO:106)；
(54) the PCR reaction primers for detecting MYC genes have the following primer sequences:
5'-TCAAGAGGCGAACACACAAC-3'(SEQ ID NO:107)；
5'-TGGACGGACAGGATGTATGC-3'(SEQ ID NO:108)；
(55) PCR primers for detecting MYOD1 gene have the following primer sequences:
5'-GGACGTGCCTTCTGAGTC-3'(SEQ ID NO:109)；
5'-GCACCTGGTATATCGGGTTG-3'(SEQ ID NO:110)；
(56) the primer sequence of the PCR reaction primer for detecting the NF1 gene is as follows:
5'-GAATCATCACCAATTCCGCA-3'(SEQ ID NO:111)；
5'-CACAACCTTGCACTGCTTTAT-3'(SEQ ID NO:112)；
(57) PCR reaction primers for detecting NFKB1 gene have the following primer sequences:
5'-GTGCGGCTCATGTTTACAG-3'(SEQ ID NO:113)；
5'-ATGGCGTCTGATACCACGG-3'(SEQ ID NO:114)；
(58) the PCR reaction primer for detecting the PCNA gene has the following primer sequence:
5'-CACTAAGGGCCGAAGATAACG-3'(SEQ ID NO:115)；
5'-CAGCATCTCCAATATGGCTGA-3'(SEQ ID NO:116)；
(59) the PCR reaction primer for detecting the PIDD gene has the following primer sequence:
5'-TCACCCACCTGTACGCAC-3'(SEQ ID NO:117)；
5'-AGAGCGATGAGGTTCACAC-3'(SEQ ID NO:118)；
(60) the PCR reaction primers for detecting the PPM1D gene have the following primer sequences:
5'-TGGTCATCATTCGGGGCAT-3'(SEQ ID NO:119)；
5'-ATCCTTCGGGTCATCCTGAA-3'(SEQ ID NO:120)；
(61) the PCR reaction primers for detecting the PRC1 gene have the following primer sequences:
5'-AGTGGAATTGATGCGAAAACAG-3'(SEQ ID NO:121)；
5'-CTCACGCCTAGAAGCCTTTG-3'(SEQ ID NO:122)；
(62) the PCR reaction primer for detecting PRKCA gene has the following primer sequence:
5'-TGTCACAGTACGAGATGCAAAA-3'(SEQ ID NO:123)；
5'-CTTTCATTCTTGGGATCAGGAA-3'(SEQ IDNO:124)；
(63) the primer sequence of the PCR reaction primer for detecting the PTEN gene is as follows:
5'-GGGACGAACTGGTGTAATGA-3'(SEQ ID NO:125)；
5'-TGGTCCTTACTTCCCCATAGAA-3'(SEQ ID NO:126)；
(64) the PCR reaction primers for detecting the PTTG1 gene have the following primer sequences:
5'-CCCGTGTGGTTGCTAAGG-3'(SEQ ID NO:127)；
5'-CGTGGTGTTGAAACTTGAGAT-3'(SEQ ID NO:128)；
(65) the PCR reaction primers for detecting the RB1 gene have the following primer sequences:
5'-ACTTTGTGAACGCCTTCTGT-3'(SEQ ID NO:129)；
5'-ACGTTTGAATGTCTCCTGAACA-3'(SEQ ID NO:130)；
(66) the primer sequence of the PCR reaction primer for detecting the RELA gene is as follows:
5'-TGGGGACTACGACCTGAATG-3'(SEQ ID NO:131)；
5'-GGGCACGATTGTCAAAGATG-3'(SEQ ID NO:132)；
(67) the PCR reaction primers for detecting the SESN2 gene have the following primer sequences:
5'-CTTACCTGGTAGGCTCCCAC-3'(SEQ ID NO:133)；
5'-GCAACTTGTTGATCTCGCTG-3'(SEQ ID NO:134)；
(68) the PCR reaction primers for detecting the SIAH1 gene have the following primer sequences:
5'-CCATTCGCAACTTGGCTATG-3'(SEQ ID NO:135)；
5'-GGCACGGACAGGAATAAGG-3'(SEQ ID NO:136)；
(69) the PCR reaction primers for detecting the SIRT1 gene have the following primer sequences:
5'-GTGTCATAGGTTAGGTGGTGA-3'(SEQ ID NO:137)；
5'-GCCAATTCTTTTTGTGTTCGTG-3'(SEQ ID NO:138)；
(70) the PCR reaction primer for detecting the STAT1 gene has the following primer sequence:
5'-GGCTGAATTTCGGCACCT-3'(SEQ ID NO:139)；
5'-GGCTGAATTTCGGCACCT-3'(SEQ ID NO:140)；
(71) the PCR reaction primers for detecting the TADA3 gene have the following primer sequences:
5'-CTGATGACCCTATCGACGTG-3'(SEQ ID NO:141)；
5'-CAGGGGTGGGATCTTGTAAT-3'(SEQ ID NO:142)；
(72) the PCR reaction primer for detecting TNF gene has the following primer sequence:
5'-AGGCCAAGCCCTGGTATG-3'(SEQ ID NO:143)；
5'-GGGCCGATTGATCTCAGC-3'(SEQ ID NO:144)；
(73) the PCR reaction primers for detecting the TNFRSF10B gene have the following primer sequences:
5'-CAGTTGCAGCCGTAGTCTTG-3'(SEQ ID NO:145)；
5'-CAGGTCGTTGTGAGCTTCT-3'(SEQ ID NO:146)；
(74) the PCR reaction primers for detecting the TNFRSF10D gene have the following primer sequences:
5'-TTGGCTTTTCATGTCGGAAGA-3'(SEQ ID NO:147)；
5'-CCAGGAACTCGTGAAGGAC-3'(SEQ ID NO:148)；
(75) the primer sequence of the PCR reaction primer for detecting the TP53 gene is as follows:
5'-CAGCTTTGAGGTGCGTGTTT-3'(SEQ ID NO:149)；
5'-CCTTTCTTGCGGAGATTCTCT-3'(SEQ ID NO:150)；
(76) the primer sequence of the PCR reaction primer for detecting the TP53AIP1 gene is as follows:
5'-CCTGGCTGGGTAAGTCCC-3'(SEQ ID NO:151)；
5'-TCCTGGTGAGTCTGAAAACTTG-3'(SEQ ID NO:152)；
(77) the primer sequence of the PCR reaction primer for detecting the TP53BP2 gene is as follows:
5'-AAGTGCGTCCGTTCTCAATG-3'(SEQ ID NO:153)；
5'-GTTCTTCCTCAGAGTACCAAAG-3'(SEQ ID NO:154)；
(78) the primer sequence of the PCR reaction primer for detecting the TP63 gene is as follows:
5'-TCATTTGATTCGAGTAGAGGGG-3'(SEQ ID NO:155)；
5'-TGGGGTGGCTCATAAGGT-3'(SEQ ID NO:156)；
(79) the primer sequence of the PCR reaction primer for detecting the TP73 gene is as follows:
5'-ACGAGGACACGTACTACCTT-3'(SEQ ID NO:157)；
5'-TGCCGATAGGAGTCCACCA-3'(SEQ ID NO:158)；
(80) the primer sequence of the PCR reaction primer for detecting the TRAF2 gene is as follows:
5'-CTCATGCTGACCGAATGTC-3'(SEQ ID NO:159)；
5'-CCGTCACAAGTTAAGGGGAA-3'(SEQ ID NO:160)；
(81) the PCR reaction primers for detecting the TSC1 gene have the following primer sequences:
5'-TGGCGGAAGTCTATCTCGTC-3'(SEQ ID NO:161)；
5'-CAAGGGTACATTCCATAAAGGC-3'(SEQ ID NO:162)；
(82) the primer sequence of the PCR reaction primer for detecting the WT1 gene is as follows:
5'-TGACTTCAAGGACTGTGAACG-3'(SEQ ID NO:163)；
5'-GGGAGAACTTTCGCTGACAA-3'(SEQ ID NO:164)；
(83) the PCR reaction primers for detecting the XRCC5 gene have the following primer sequences:
5'-GACTTCCTGGATGCACTAATCG-3'(SEQ ID NO:165)；
5'-CTAAGCGAAAGGGGCCAT-3'(SEQ ID NO:166)；
(84) the PCR reaction primer for detecting the RPRM gene has the following primer sequence:
5'-TGAACGCGACCTTCCTGAA-3'(SEQ ID NO:167)；
5'-ACCACGGTAAGCGACAG-3'(SEQ ID NO:168)；
(85) the PCR reaction primers for detecting the ACTB gene have the following primer sequences:
5'-ATGTACGTTGCTATCCAGGC-3'(SEQ ID NO:169)；
5'-TCCTTAATGTCACGCACGAT-3'(SEQ ID NO:170)；
(86) the PCR reaction primers for detecting the B2M gene have the following primer sequences:
5'-AGGCTATCCAGCGTACTCCA-3'(SEQ ID NO:171)；
5'-GGCAGGCATACTCATCTTTT-3'(SEQ ID NO:172)；
(87) the PCR reaction primers for detecting the GAPDH gene have the following primer sequences:
5'-TGGGCTACACTGAGCACC-3'(SEQ ID NO:173)；
5'-AGTGGTCGTTGAGGGCAATG-3'(SEQ ID NO:174)；
(88) the PCR reaction primers for detecting the HPRT1 gene have the following primer sequences:
5'-AAAAGGACCCCACGAAGTGT-3'(SEQ ID NO:175)；
5'-GTCAAGGGCATATCCTACAACA-3'(SEQ ID NO:176)；
(89) the PCR reaction primers for detecting the RPLP0 gene have the following primer sequences:
5'-AGATTGGCTACCCAACTGTT-3'(SEQ ID NO:177)；
5'-GAAGGTGTAATCCGTCTCCAC-3'(SEQ ID NO:178)。
the invention also provides application of the PCR reagent in preparation of a kit for detecting human oral squamous carcinoma cells and normal oral epithelial cells.
Further: the kit adopts a real-time fluorescent quantitative PCR method for detection.
Further: the reaction system of the real-time fluorescent quantitative PCR is as follows: 10 mul of Taq polymerase reaction mixture, 0.1 mul-0.2 mul of upstream primer, 0.1 mul-0.2 mul of downstream primer, 25ng-100ng of cDNA template, and sterile distilled water to make up to 20 mul.
Further, the Taq polymerase is 2 × from TaKaRa
Figure BDA0002580723320000081
Premix Ex Taq polymerase.
Further: the reaction procedure of the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 ℃ for 5 seconds, 59 ℃ for 20 seconds, 40 cycles. The dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.
The PCR reaction instrument is LightCycler480 of Roche. The 89 pairs of primers were placed in a 96-well plate for real-time fluorescent quantitative PCR detection. Changes in the core molecule of the tumor core molecule can be obtained by a single reaction.
Compared with the prior art, the invention has the advantages and positive effects that: the invention provides a reagent for detecting the change of a P53 signal channel core molecule, which can rapidly detect genes related to tumor occurrence at a transcription level through real-time fluorescence quantification by virtue of the detection reagent, and analyzes and researches a P53 signal channel core molecule and an action mechanism in a tumor cell on the basis, thereby rapidly and accurately finding a core regulation signal channel of a tumor-related drug, and providing a powerful tool for screening an anti-cancer drug, researching a mechanism of a new targeted drug and the like.
The invention determines 84 genes related to the P53 signal path through a large number of creative experiments, the invention focuses on the combination of primers, and experiments prove that the conditions of primer dimer and non-specific amplification during PCR amplification can be avoided only through proper primer combination, thereby ensuring the effective amplification of all target genes. The primer dimer often appears in the combination of any primer during the specific PCR, which affects the failure of the target gene amplification, or the non-specific amplification, which greatly affects the effectiveness of the PCR result. Under the same experimental condition, PCR detection of 89 genes (84 of which are detection genes and 5 of which are reference genes) is carried out simultaneously, so that not only is primer design required, but also the optimal amplification condition is required to be well grasped, which is a technical difficulty. The most central is primer design, the invention obtains the best amplification condition through multiple pcr and adjustment, and ensures that the amplification efficiency of each gene is very high without nonspecific amplification.
The invention also provides a PCR method for detection, the experimental system can be carried out on any real-time fluorescence quantitative PCR instrument, the experimental operation is simple and convenient, the cost is low, the result repeatability is good, the downstream unnecessary detection is reduced, and the PCR method is an important means for researching the action mechanism of the tumor-related medicine.
Other features and advantages of the present invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
Drawings
FIG. 1 shows that the P53 signal channel is utilized to screen the genes differentially expressed by human oral squamous carcinoma cell SCC-25 and normal oral epithelial cells, wherein 1: squamous cell carcinoma SCC-25 cells of the oral cavity; 2: normal oral epithelial cells.
FIG. 2 shows the product dissolution curve analysis of real-time fluorescent quantitative PCR.
FIG. 3 shows the screening results of P53 signaling pathway core molecule.
FIG. 4 shows the expression results of real-time fluorescence PCR detection of core molecules.
FIG. 5 shows flow cytometry to detect the effect of RPRM on SCC-25 cell cycle.
FIG. 6 shows Western blotting detection of the regulation of RPRM on core protein.
FIG. 7 shows flow cytometry detection of apoptosis of SCC-25 cells.
FIG. 8 shows the regulation of apoptosis core protein by PIDD detected by Western blotting.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.
Example 1
The invention is exemplified by human oral squamous carcinoma SCC-25 and human oral normal epithelial cells, but the invention is not limited to the two cells, and the detection reagent can be applied to tumor cell lines including liver cancer, gastric cancer and the like, and can also be used for detecting clinical tumor tissues, tissues beside the cancer and the like.
The invention is characterized by culturing two cell lines: human oral squamous carcinoma SCC-25, human oral normal epithelial cells were then tested for changes in core molecules in both cell lines using P53 signaling pathway molecular detection reagents.
Human oral squamous carcinoma SCC-25, human oral normal epithelial cells HOK purchased from ATCC company, DMEM medium, fetal bovine serum purchased from Gibco company, RNAiso for total RNA extraction, reverse transcription kit, 2 ×
Figure BDA0002580723320000091
The Premix Ex Taq kit was purchased from TaKaRa.
The method for detecting the P53 signal channel core molecule by using the reagent comprises the following specific steps:
culture and passage of human oral squamous carcinoma SCC-25 and human oral normal epithelial cell HOK
Taking SCC-25 cells as an example, 1mL of 0.2% pancreatin solution is added into a 60mm cell culture plate, the pancreatin is gently shaken to be in full contact with the cells, the cell culture plate is incubated for 2min at 37 ℃ in a carbon dioxide incubator, after most of the cells are observed to float under a mirror, 4mL of DMEM medium containing 10% FBS is added, the DMEM medium is evenly blown and added into a 15mL centrifugal tube, the 15mL centrifugal tube is centrifuged for 6min at 2000g, the supernatant is discarded, a corresponding appropriate volume of culture solution is added according to the cell type, and the cell is blown and beaten repeatedly by a 10mL pipette for a plurality of times to free. In this process, air bubbles are prevented from being generated, so as not to influence the cell count. Taking 20 mu L of cell suspension, dripping into a cell counting plate, and counting the total number of cells in four big grids at four corners according to a cell counting method. When counting, only cells with intact cell morphology are counted, and the number of stacked cells is counted as 1 cell. The calculation formula is as follows:
cell concentration (one/mL) ═ × 10 (total number of 4 large cells)4× dilution factor/4.
Diluting with corresponding culture solution cell suspension to 5 × 10 according to cell type6Adding 2mL of cell dilution into 60mm cell culture dish, placing at 37 deg.C and 5% CO2Culturing in a cell culture box, wherein the cells adhere to the wall after 0.5-1 h generally, grow after several hours, and become single cells after 24-48 hLayer cells.
Second, extraction of Total RNA
1) Taking CO2After sucking out the culture medium from a disk of cells in a 60mm plate in logarithmic growth phase in an incubator, washing the disk of cells for 2 times by using sterile 1 × PBS, adding 1mL of RNAasso on the surfaces of the cells, repeatedly sucking the cells uniformly by using a gun head, and transferring the cells into a sterile tube.
2) Incubate at room temperature for 5min to solubilize the nucleoprotein. Add 0.2mL phenol/chloroform (1:1), shake vigorously for 15sec, incubate at room temperature for 2-3 min.
3) Cells were stratified by centrifugation at 12000g for 15min at 4 ℃.
4) Transferring the upper layer liquid to a centrifuge tube without RNase, adding 0.5mL isopropanol, reversing and mixing uniformly for 3 times, and incubating for 10min at room temperature.
5) Centrifugation at 12000g for 10min at 4 ℃ resulted in the formation of a bottom pellet of RNA.
6) The supernatant was discarded and the pellet was washed with 1mL of RNase-free 75% ethanol. Centrifuge at 7000g for 5min at 4 ℃.
8) The RNA was air dried for about 5-10 min. It should not be dried too much.
9) RNA was dissolved in 30-50. mu.L DEPC water.
The results are shown in FIG. 1, and it can be seen from the electrophoresis in the figure that lane 1 is Huh7, lane 2 is normal liver cells, the total RNA band is complete, and it is suitable for downstream reverse transcription and other experiments.
Synthesis of cDNA
1) The following reaction mixture was added to a pre-cooled tube:
1-5 mug of total RNA;
oligo(dT)(0.5μg/μl) 1μL；
the volume was adjusted to 12. mu.L with RNase-free water.
2) Mixing, and centrifuging for 3-5 sec.
3) Acting at 70 deg.C for 5min, ice-cooling for 30sec, and centrifuging for 3-5 sec.
4) The tube was ice-cooled and the following components were added:
5×Reaction buffer 4μL；
Rnase Inhibiter(20U/μL) 1μL；
dNTP mix(10mM) 2μL；
5) mix gently and centrifuge for 3-5 sec.
6) After being bathed at 37 ℃ for 5min, 1. mu.L of M-MuLV Reverse Transcriptase (20U/. mu.L) was added and mixed well.
7) Acting at 37 deg.C for 60 min. The reaction was terminated by incubation at 70 ℃ for 10min and downstream testing was performed on ice.
Four, real-time fluorescent quantitative PCR
Taking the synthesized cDNA as a template, and carrying out real-time fluorescence quantitative PCR reaction according to the following system:
Figure BDA0002580723320000111
the following program was set up on the LightCycler480 instrument from roche: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 ℃ for 5 seconds, 59 ℃ for 20 seconds, 40 cycles. The dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.
The upstream and downstream primers in the PCR reaction are respectively the PCR reagents (including 84 primers of the detection genes and 5 primers of the reference genes) described in the invention, and the sequences are shown as SEQ ID NO:1-SEQ ID NO: 178.
And (3) placing the primers on a 96-well plate to perform PCR reaction, wherein each pair of primers is placed in any one well, and 89 pairs of primers are placed in 89 wells in total.
FIG. 2 shows the analysis of the dissolution curve of human oral squamous carcinoma SCC-25 and human oral normal epithelial cell HOK after real-time fluorescence quantitative PCR, and the results in FIG. 2 show that the dissolution curve of the target gene is a single peak and has no miscellaneous peak, which indicates that the amplification is good and is suitable for downstream Ct value analysis.
Fifth, data analysis
And calculating the expression difference of the tumor core genes by using the Ct value of each gene.
FIG. 3 shows the results of differential expression analysis after real-time fluorescent quantitative PCR of human oral squamous carcinoma SCC-25 and human oral normal epithelial cells HOK, from which core molecules related to the P53 signal pathway, mainly including RPRM gene, PIDD gene, STAT1 gene, CHEK1 gene and CDKN2A gene, can be screened out.
Sixthly, real-time fluorescence quantitative PCR verification of screening P53 signal channel core molecule
The screened core genes are subjected to re-detection of real-time fluorescence PCR by adopting the method, the expression of the target gene is detected by adopting the average value of the real-time fluorescence PCR, the result is shown in figure 4, the expressions of the RPRM gene, the PIDD gene, the STAT1 gene, the CHEK1 gene and the CDKN2A gene are obviously changed, and the screened RPRM gene and the PIDD gene are used as candidate molecules for verifying the downstream P53 signal channel function.
Seventhly, detecting the influence of RPRM on the tumor cell cycle by adopting a flow cytometer
1. Preparation of cell samples:
after culturing the SCC-25 cells treated with the RPRM overexpression vector and the control cells for 24 hours, cell cycle measurements were performed. The culture broth was carefully collected into a centrifuge tube for use. Digesting the cells with pancreatin until the cells can be blown down by a pipette or a gun head, adding the previously collected cell culture solution, blowing down all adherent cells, and blowing off the cells gently. Again collected in the centrifuge tube. Centrifuging at about 1000g for 3-5 min to precipitate cells. The supernatant was carefully aspirated, and about 50. mu.l of the culture medium was left to avoid aspiration of the cells. Approximately 1ml of ice-cooled PBS was added, the cells were resuspended, and transferred to a 1.5 ml centrifuge tube. The cells were pelleted by centrifugation again and the supernatant carefully aspirated, approximately 50 microliters or so of PBS could remain to avoid aspiration of the cells. Gently flick the bottom of the centrifuge tube to properly disperse the cells and avoid cell clumping.
2. Cell fixation:
adding into 1ml ice bath precooled 70% ethanol, lightly blowing and mixing evenly, and fixing for 30 minutes or longer at 4 ℃. Generally, a dyeing effect can be better ensured after 2 hours or more of fixation, and the effect can be better after 12-24 hours of fixation. Centrifuging at about 1000g for 3-5 min to precipitate cells. The supernatant was carefully aspirated, and about 50. mu.l or so of 70% ethanol remained to avoid aspiration of the cells. Approximately 1ml of ice-cold PBS was added to resuspend the cells. The cells were pelleted by centrifugation again and the supernatant carefully aspirated, approximately 50 microliters or so of PBS could remain to avoid aspiration of the cells. Gently flick the bottom of the centrifuge tube to properly disperse the cells and avoid cell clumping.
3. Preparation of an propidium iodide staining solution: referring to table 1, an appropriate amount of propidium iodide staining solution was prepared according to the number of samples to be detected:
TABLE 1 preparation of propidium iodide staining solution
1 sample 6 samples 12 samples
Staining buffer 0.5ml 3ml 6ml
Propidium iodide staining solution (20X) 25μl 150μl 300μl
RNase A(50X) 10μl 60μl 120μl
Final volume 0.535ml 3.21ml 6.42ml
4. Dyeing: 0.5ml of propidium iodide staining solution was added to each tube of cell sample, and the cell pellet was slowly and sufficiently resuspended and incubated at 37 ℃ in the dark for 30 minutes. Subsequently, the cells can be stored at 4 ℃ or in an ice bath protected from light. After the staining is finished, the flow detection is preferably finished within 24 hours, and the flow detection can be preferably finished on the same day.
5. Flow detection and analysis: red fluorescence was detected with a BD Calibur flow cytometer at the 488nm excitation wavelength, while light scattering was detected. Cellular DNA content analysis and light scattering analysis were performed using FlowJo software.
6. Interpretation of results
After the RPRM is over-expressed, the gene is found to be capable of obviously inhibiting the SCC-25 cell cycle (the result is shown in figure 5).
Eighthly, detecting the influence of RPRM on SCC-25 cell cycle related gene by Western blotting
1. Collecting protein samples
Adherent cells were lysed using RIPA lysate as required for the experiment, and protease inhibitors, phosphatase inhibitors or deacetylase inhibitor cocktail were additionally added to prevent degradation of proteins.
After the protein samples are collected, the protein concentration of each protein sample needs to be determined in order to ensure that the loading amount of each protein sample is consistent. Protein concentration was detected using the BCA protein concentration assay kit.
2. Electrophoresis
Preparing polyacrylamide gel, 12% of separation gel and 5% of lamination gel.
100ml of Bio-Rad 10 Xelectrophoresis buffer was dissolved in 900ml of distilled water to prepare 1L of electrophoresis buffer, which was prepared as it is.
1 Xbuffer (10 Xmembrane buffer stock 100ml +200ml methanol (analytically pure) +700ml deionized water) was prepared; 100ml 10 × TBS buffer from Biochemical company was dissolved in 900ml distilled water to prepare 1L 1 × TBS buffer, and 1ml Tween-20 was added to prepare 1 × TBST buffer.
0.5ml of skimmed milk and 9.5ml of TBST solution are prepared into a confining liquid containing 5 percent of skimmed milk, and the confining liquid is prepared for use.
0.5ml of skimmed milk and 9.5ml of TBST are prepared into an antibody diluent containing 5% of skimmed milk, wherein the primary antibody diluent contains 0.002g of sodium azide, and the secondary antibody diluent does not contain, so that the antibody diluent is prepared for use.
And putting the prefabricated gel into an electrophoresis device, immersing the electrophoresis device in 1x electrophoresis liquid, and checking to determine that no liquid leakage exists.
The power supply is switched on, and the electrophoresis is carried out for 15min at a constant voltage of 80V.
Add 20. mu.g of protein to the loading well and electrophoresis marker to the very edge of the lane.
The power is switched on, and the electrophoresis is carried out for 30min at a constant voltage of 80V and then for 1h at 120V.
And finishing electrophoresis when the lowest color band on the prefabricated glue is 1-2cm away from the lower edge of the glue, and preparing to transfer the film.
3. Rotary film
A PVDF membrane of the same size as the gel was cut out and soaked in anhydrous methanol for 10 seconds.
And sealing the rotary template and then placing the rotary template into a rotary film groove. Adding about 1000ml of pre-cooled membrane transferring liquid with 4 ℃ into the membrane transferring groove, and putting an ice bag into the membrane transferring groove to maintain the low temperature of the whole process.
The electrophoresis tank was placed in ice cubes. And (5) rotating the membrane for 90min at a constant current of 0.2A.
4. Sealing of
Immersing PVDF membrane in 5% skimmed milk, and shaking for 90min at room temperature.
5. Hybridization of
The blocked PVDF membrane was washed with PBST and immersed in primary antibody overnight in a shaker at 4 ℃ for 30min the next day at room temperature. PBST was washed 3 times for 15min each. The secondary antibody was immersed for 1h and washed 3 times with PBST, 15min each time.
6. Development
According to the ECL instruction, the solution A and the solution B are added into a 15ml centrifuge tube according to the proportion of 1:1, mixed by turning upside down for several times, and kept standing for 5min in a dark place. And (3) uniformly dripping the ECL mixed solution on the membrane (preferably 10001 mixed solution per membrane), sealing the membrane by using a preservative film, and fixing the membrane in a dark box. The film was quickly covered on top of the ECL film in a dark room, the cassette was closed and exposed to light according to the intensity of the fluorescence seen (1s-30 min).
Taking out the film, immediately and completely immersing the film into the developing solution for 1min, rinsing the film for 30s with clear water, immersing the film into the fixing solution, completely fixing the film after about 30s, rinsing the film with clear water, drying the film, calibrating the size of the molecular weight of the strip according to the position of a Marker, and analyzing and scanning the film.
As shown in the figure 6, the result of Western blot detection of the expression of Cyclin B1 under the treatment of RPRM shows that the expression of Cyclin B1 is obviously reduced after the RPRM is over-expressed. The result further proves that the protein related to the P53 signal path obtained by the screening of the invention has obvious specificity.
Seventhly, detecting the influence of PIDD on oral squamous cell carcinoma apoptosis by adopting flow cytometry
The total amount of the protein is divided into two groups, namely a pcDNA Control group (OE-Control) and an overexpression PIDD group (OE-PIDD)
Collecting cells: taking a proper amount of SCC-25 cells in the logarithmic growth phase, inoculating the cells into a 6-well plate, treating the cells for a corresponding time under corresponding conditions (such as the above various treatment groups), collecting the cells, and taking care to combine the supernatant and the digested cells;
and (3) cleaning cells: washing 2 times with pre-cooled PBS;
grouping: each experiment is divided into a non-dyeing group, a single-dyeing Annexin V group, a single-dyeing PI group and a PI and Annexin V double-dyeing group, and the treatment group is subjected to double-dyeing from low to high;
dyeing: the 4 Xbinding buffer was diluted to 1 Xbuffer with PBS, the residual PBS in the centrifuge tube was aspirated, 100. mu.L of 1 Xbinding buffer was added to each tube, the cells were blown with a pipette to resuspend them thoroughly, and the dye was added in the dark. Adding Annexin V or PI 5 muL into a single-dyeing group, adding Annexin V and PI 5 muL into a double-dyeing group, and gently mixing by using a pipette gun;
and (3) computer detection: after incubation for 15 minutes at room temperature in the dark, 300. mu.L of binding buffer was added and mixed well, and the cell suspension was transferred to a 5mL flow tube in the dark for 1h on-machine detection on a FACSCalibur flow cytometer.
As a result, as shown in FIG. 7, the over-expressed PIDD group (OE-PIDD) significantly increased the apoptosis of SCC-25, as compared to the pcDNA Control group (OE-Control).
Eighthly, detecting the influence of PIDD on SCC-25 apoptosis related gene by Western blotting
1. Collecting protein samples
Adherent cells were lysed using RIPA lysate as required for the experiment, and protease inhibitors, phosphatase inhibitors or deacetylase inhibitor cocktail were additionally added to prevent degradation of proteins.
After the protein samples are collected, the protein concentration of each protein sample needs to be determined in order to ensure that the loading amount of each protein sample is consistent. Protein concentration was detected using the BCA protein concentration assay kit.
2. Electrophoresis
Preparing polyacrylamide gel, 12% of separation gel and 5% of lamination gel.
100ml of Bio-Rad 10 Xelectrophoresis buffer was dissolved in 900ml of distilled water to prepare 1L of electrophoresis buffer, which was prepared as it is.
1 Xbuffer (19 Xmembrane buffer stock 100ml +200ml methanol (analytically pure) +700ml deionized water) was prepared; 100ml 10xTBS buffer from Biochemical company was dissolved in 900ml distilled water to prepare 1L of 1 xTBS buffer, and 1ml Tween-20 was added to prepare 1 xTBST buffer.
0.5ml of skimmed milk and 9.5ml of TBST solution are prepared into a confining liquid containing 5 percent of skimmed milk, and the confining liquid is prepared for use.
0.5ml of skimmed milk and 9.5ml of TBST are prepared into an antibody diluent containing 5% of skimmed milk, wherein the primary antibody diluent contains 0.002g of sodium azide, and the secondary antibody diluent does not contain, so that the antibody diluent is prepared for use.
And putting the prefabricated gel into an electrophoresis device, immersing the electrophoresis device in 1x electrophoresis liquid, and checking to determine that no liquid leakage exists.
The power supply is switched on, and the electrophoresis is carried out for 15min at a constant voltage of 80V.
Add 20. mu.g of protein to the loading well and electrophoresis marker to the very edge of the lane.
The power is switched on, and the electrophoresis is carried out for 30min at a constant voltage of 80V and then for 1h at 120V.
And finishing electrophoresis when the lowest color band on the prefabricated glue is 1-2cm away from the lower edge of the glue, and preparing to transfer the film.
3. Rotary film
A PVDF membrane of the same size as the gel was cut out and soaked in anhydrous methanol for 10 seconds.
And sealing the rotary template and then placing the rotary template into a rotary film groove. Adding about 1000ml of pre-cooled membrane transferring liquid with 4 ℃ into the membrane transferring groove, and putting an ice bag into the membrane transferring groove to maintain the low temperature of the whole process.
The electrophoresis tank was placed in ice cubes. And (5) rotating the membrane for 90min at a constant current of 0.2A.
4. Sealing of
Immersing PVDF membrane in 5% skimmed milk, and shaking for 90min at room temperature.
5. Hybridization of
The blocked PVDF membrane was washed with PBST and immersed in primary antibody overnight in a shaker at 4 ℃ for 30min the next day at room temperature. PBST was washed 3 times for 15min each. The secondary antibody was immersed for 1h and washed 3 times with PBST, 15min each time.
6. Development
According to the ECL instruction, the solution A and the solution B are added into a 15ml centrifuge tube according to the proportion of 1:1, mixed by turning upside down for several times, and kept standing for 5min in a dark place. And (3) uniformly dripping the ECL mixed solution on the membrane (preferably 1000 microliter of the mixed solution per membrane), sealing the membrane by using a preservative film, and fixing the membrane in a dark box. The film was quickly covered on top of the ECL film in a dark room, the cassette was closed and exposed to light according to the intensity of the fluorescence seen (1s-30 min).
Taking out the film, immediately and completely immersing the film into the developing solution for 1min, rinsing the film for 30s with clear water, immersing the film into the fixing solution, completely fixing the film after about 30s, rinsing the film with clear water, drying the film, calibrating the size of the molecular weight of the strip according to the position of a Marker, and analyzing and scanning the film.
As shown in FIG. 8, the expression of Casp3 in PIDD treatment is detected by Western blotting, and the result shows that after PIDD is over-expressed, the expression of Casp3 in a splicing body is obviously increased. The result further proves that the protein related to the P53 signal path obtained by the screening of the invention has obvious specificity of the P53 signal path. The high efficiency of the technical scheme of the invention is proved.
The invention concentrates the molecules closely related to the tumor core molecules on a flat plate, reacts the survival state of cells by carrying out a real-time fluorescent quantitative PCR reaction, discusses the possible way of the P53 signal channel core molecules in tumor/normal cells, and provides the most direct evidence for researching the regulation and control of key proteins; the invention can quickly and accurately find the related molecules of the tumor core molecules from the transcription level, and provides a powerful tool for screening anti-cancer drugs, discussing the mechanism of new targeted drugs and the like.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
agtctggctc gttctcagtg 20
<210>21
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
tgttacaaat cacccctcaa gg 22
<210>22
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
cctgatactt ttctggatgc c 21
<210>23
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
gcctgaaaac cagatgacta tc 22
<210>24
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
ggccagcaaa cttccgttta 20
<210>25
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
ctgtgggtgg acccctat 18
<210>26
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
gcctcctcgt acaagacg 18
<210>27
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
gctgttgttg agcgaattgt 20
<210>28
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
gcaagttgag gagttccaca 20
<210>29
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
tgtctacggc acagatggat 20
<210>30
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
ggactcgtct tcaggggaa 19
<210>31
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
actttcgcct gagcctattt t 21
<210>32
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
tggtctgact gcttgctctt 20
<210>33
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
ctcaacgtgc aagcctcg 18
<210>34
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
ctcaagaaag tgctgatccc 20
<210>35
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
agtctgcaca cgataatggc 20
<210>36
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
tgcttgggct gtaccttca 19
<210>37
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
ggcacttgaa cagatactgg a 21
<210>38
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
caatatggga tagcgggtct 20
<210>39
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
tcctcttttt acaccccagt ca 22
<210>40
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
cggttgtcaa ggtttgtagt tc 22
<210>41
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
tgacaatgga aacttggtgg ac 22
<210>42
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
gagcgatata ggccacttct g 21
<210>43
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
gatgtgctta tgcaggattc c 21
<210>44
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
atgtactgac caggagggat ag 22
<210>45
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
tggtgtttga gcatgtagac c 21
<210>46
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
atccttgatc gtttcggctg 20
<210>47
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
gatggaactt cgactttgtc a 21
<210>48
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
cacaagggta caagacagtg 20
<210>49
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
tggagccttc ggctgact 18
<210>50
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
taactattcg gtgcgttggg 20
<210>51
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
cttactgcaa tgctcgctgg 20
<210>52
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
tgaggggttt gttgtaccat c 21
<210>53
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
tatctgcctt agtgggtatc ca 22
<210>54
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
tgtcgtaaaa cgtgcctttg 20
<210>55
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
atcagaccgg cagattaacc 20
<210>56
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
ttggccttac agcggtagat 20
<210>57
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
ctagccccag gattacaagg 20
<210>58
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
ctcatccgat ttggctcttt c 21
<210>59
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
atcccaggag gtcacttctg 20
<210>60
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
acaacagcgg ttcttgctc 19
<210>61
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
aagcccctcc agaaacaaga 20
<210>62
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
cttgggtact tgccaaatgt 20
<210>63
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
tgccgcaaag tgtgtaacg 19
<210>64
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
tgccgcaaag tgtgtaacg 19
<210>65
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>65
cccctctgtc tactattaag gc 22
<210>66
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>66
gggactggta gctggtattg 20
<210>67
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>67
tcggtaacag cccgaattat c 21
<210>68
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>68
cactgaaaat tgacgtgagg aa 22
<210>69
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>69
aaaggtggga tacgaaaaga cc 22
<210>70
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>70
ctgttcttct tagagcgttt ga 22
<210>71
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>71
tggctgacct ggtacaagag 20
<210>72
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>72
gtagatgcgt ctgagttcca t 21
<210>73
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>73
gattgtgtga tgaaggacat gg 22
<210>74
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>74
gttgctggtg agtgtgcatt 20
<210>75
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>75
tttaacaggc aagtccaact ca 22
<210>76
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>76
gccacccttc ttatacttca ct 22
<210>77
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>77
cggctgactg atatggcag 19
<210>78
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>78
gtgatgttat ccagcaggtc 20
<210>79
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>79
cctgatccag gcgttttg 18
<210>80
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>80
atccatgtag cgactttccc 20
<210>81
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>81
tctttgcctt actcctagcc a 21
<210>82
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>82
gcgcctaata tgatacggac a 21
<210>83
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>83
gccctcacaa agccaatg 18
<210>84
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>84
tgcttgctgt actccgaca 19
<210>85
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>85
tgaccaggag attgacatgg g 21
<210>86
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>86
aaccgcatca ggacctca 18
<210>87
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>87
tgctgacctc tgttacctct 20
<210>88
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>88
gcttattccc cacaatgtag tt 22
<210>89
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>89
ctgaaccttc caaagatggc 20
<210>90
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>90
tcaccaggca agtctcctca 20
<210>91
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>91
acaggtggca cagcttaaac 20
<210>92
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>92
aactgctgcg ttagcatgag 20
<210>93
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>93
ggaaaacctg tggttgaagg 20
<210>94
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>94
agtcttcgtt gagatggtgc 20
<210>95
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>95
agtacagtgc aatgagggac 20
<210>96
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>96
ctgagcctgt tttgtgtcta c 21
<210>97
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>97
tgcctttgtg gctaaacact 20
<210>98
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>98
gtcccgtttt gtccttacga 20
<210>99
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>99
gcaggggaga gtgatacaga 20
<210>100
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>100
aagccaattc tcacgaaggg 20
<210>101
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>101
tttgatccct gcaactcagt g 21
<210>102
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>102
tctcgtggtc ttttctcaca t 21
<210>103
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>103
caaacccctg tccagtcag 19
<210>104
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>104
tgggagttca agcatctcct 20
<210>105
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>105
gtcagagccc ttaacctttt tc 22
<210>106
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>106
agaggctgct taatccactg 20
<210>107
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>107
tcaagaggcg aacacacaac 20
<210>108
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>108
tggacggaca ggatgtatgc 20
<210>109
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>109
ggacgtgcct tctgagtc 18
<210>110
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>110
gcacctggta tatcgggttg 20
<210>111
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>111
gaatcatcac caattccgca 20
<210>112
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>112
cacaaccttg cactgcttta t 21
<210>113
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>113
gtgcggctca tgtttacag 19
<210>114
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>114
atggcgtctg ataccacgg 19
<210>115
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>115
cactaagggc cgaagataac g 21
<210>116
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>116
cagcatctcc aatatggctg a 21
<210>117
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>117
tcacccacct gtacgcac 18
<210>118
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>118
agagcgatga ggttcacac 19
<210>119
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>119
tggtcatcat tcggggcat 19
<210>120
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>120
atccttcggg tcatcctgaa 20
<210>121
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>121
agtggaattg atgcgaaaac ag 22
<210>122
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>122
ctcacgccta gaagcctttg 20
<210>123
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>123
tgtcacagta cgagatgcaa aa 22
<210>124
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>124
ctttcattct tgggatcagg aa 22
<210>125
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>125
gggacgaact ggtgtaatga 20
<210>126
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>126
tggtccttac ttccccatag aa 22
<210>127
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>127
cccgtgtggt tgctaagg 18
<210>128
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>128
cgtggtgttg aaacttgaga t 21
<210>129
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>129
actttgtgaa cgccttctgt 20
<210>130
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>130
acgtttgaat gtctcctgaa ca 22
<210>131
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>131
tggggactac gacctgaatg 20
<210>132
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>132
gggcacgatt gtcaaagatg 20
<210>133
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>133
cttacctggt aggctcccac 20
<210>134
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>134
gcaacttgtt gatctcgctg 20
<210>135
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>135
ccattcgcaa cttggctatg 20
<210>136
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>136
ggcacggaca ggaataagg 19
<210>137
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>137
gtgtcatagg ttaggtggtg a 21
<210>138
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>138
gccaattctt tttgtgttcg tg 22
<210>139
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>139
ggctgaattt cggcacct 18
<210>140
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>140
ggctgaattt cggcacct 18
<210>141
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>141
ctgatgaccc tatcgacgtg 20
<210>142
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>142
caggggtggg atcttgtaat 20
<210>143
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>143
aggccaagcc ctggtatg 18
<210>144
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>144
gggccgattg atctcagc 18
<210>145
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>145
cagttgcagc cgtagtcttg 20
<210>146
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>146
caggtcgttg tgagcttct 19
<210>147
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>147
ttggcttttc atgtcggaag a 21
<210>148
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>148
ccaggaactc gtgaaggac 19
<210>149
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>149
cagctttgag gtgcgtgttt 20
<210>150
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>150
cctttcttgc ggagattctc t 21
<210>151
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>151
cctggctggg taagtccc 18
<210>152
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>152
tcctggtgag tctgaaaact tg 22
<210>153
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>153
aagtgcgtcc gttctcaatg 20
<210>154
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>154
gttcttcctc agagtaccaa ag 22
<210>155
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>155
tcatttgatt cgagtagagg gg 22
<210>156
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>156
tggggtggct cataaggt 18
<210>157
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>157
acgaggacac gtactacctt 20
<210>158
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>158
tgccgatagg agtccacca 19
<210>159
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>159
ctcatgctga ccgaatgtc 19
<210>160
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>160
ccgtcacaag ttaaggggaa 20
<210>161
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>161
tggcggaagt ctatctcgtc 20
<210>162
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>162
caagggtaca ttccataaag gc 22
<210>163
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>163
tgacttcaag gactgtgaac g 21
<210>164
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>164
gggagaactt tcgctgacaa 20
<210>165
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>165
gacttcctgg atgcactaat cg 22
<210>166
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>166
ctaagcgaaa ggggccat 18
<210>167
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>167
tgaacgcgac cttcctgaa 19
<210>168
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>168
accacggtaa gcgacag 17
<210>169
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>169
atgtacgttg ctatccaggc 20
<210>170
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>170
tccttaatgt cacgcacgat 20
<210>171
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>171
aggctatcca gcgtactcca 20
<210>172
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>172
ggcaggcata ctcatctttt 20
<210>173
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>173
tgggctacac tgagcacc 18
<210>174
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>174
agtggtcgtt gagggcaatg 20
<210>175
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>175
aaaaggaccc cacgaagtgt 20
<210>176
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>176
gtcaagggca tatcctacaa ca 22
<210>177
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>177
agattggcta cccaactgtt 20
<210>178
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>178
gaaggtgtaa tccgtctcca c 21

Claims (7)

1. A PCR reagent for detecting the expression of a cell P53 signal path molecule, which is characterized by comprising the following primers:
(1) the PCR reaction primers for detecting the APAF1 gene have the following primer sequences:
5'- CACACGGTTGGATCAGGAT -3'；
5'- GGAGACGGTCTTTAGCCTCT -3'；
(2) the PCR reaction primer for detecting the ATM gene has the following primer sequence:
5'- TGATCTTGTGCCTTGGCTAC -3'；
5'- ATGGTGTACGTTCCCCATGT -3'；
(3) the PCR reaction primers for detecting the ATR gene have the following primer sequences:
5'- CCCTTGAATACAGTGGCCTA -3'；
5'- CCTTGAAAGTACGGCAGTTC -3'；
(4) the PCR reaction primer for detecting the BAI1 gene has the following primer sequence:
5'- CAACCTGGTTCTCAGCATCC -3'；
5'- GACGGTCGTGTTCCTCTG -3'；
(5) the PCR reaction primer for detecting the BAX gene has the following primer sequence:
5'- CCGAGAGGTCTTTTTCCGAG -3'；
5'- CAGCCCATGATGGTTCTGAT -3'；
(6) the PCR reaction primers for detecting the BBC3 gene have the following primer sequences:
5'- ACCTCAACGCACAGTACGAG -3'；
5'- GGAGTCCCATGATGAGATTGT -3'；
(7) the PCR reaction primers for detecting the BCL2 gene have the following primer sequences:
5'- GTGGGGTCATGTGTGTGG -3'；
5'- GGTTCAGGTACTCAGTCATCC -3'；
(8) the PCR reaction primers for detecting the BCL2A1 gene have the following primer sequences:
5'- GTGCTACAAAATGTTGCGTTC -3'；
5'- GCAATTTGCTGTCGTAGAAGTT -3'；
(9) the PCR reaction primer for detecting BID gene has the following primer sequence:
5'- TGGACCGTAGCATCCCTCC -3'；
5'- TAGGTGCGTAGGTTCTGGT -3'；
(10) the PCR reaction primers for detecting the BIRC5 gene have the following primer sequences:
5'- GGACCACCGCATCTCTACAT -3'；
5'- AGTCTGGCTCGTTCTCAGTG-3'；
(11) the PCR reaction primers for detecting BRCA1 gene have the following primer sequences:
5'- TGTTACAAATCACCCCTCAAGG-3'；
5'- CCTGATACTTTTCTGGATGCC -3'；
(12) the PCR reaction primers for detecting BRCA2 gene have the following primer sequences:
5'- GCCTGAAAACCAGATGACTATC -3'；
5'- GGCCAGCAAACTTCCGTTTA -3'；
(13) the primer sequence of the PCR reaction primer for detecting the BTG2 gene is as follows:
5'- CTGTGGGTGGACCCCTAT -3'；
5'- GCCTCCTCGTACAAGACG -3'；
(14) the PCR reaction primers for detecting the CASP2 gene have the following primer sequences:
5'-GCTGTTGTTGAGCGAATTGT -3'；
5'-GCAAGTTGAGGAGTTCCACA -3'；
(15) the PCR reaction primers for detecting the CASP9 gene have the following primer sequences:
5'- TGTCTACGGCACAGATGGAT -3'；
5'- GGACTCGTCTTCAGGGGAA -3'；
(16) the PCR reaction primers for detecting the CCNB1 gene have the following primer sequences:
5'- ACTTTCGCCTGAGCCTATTTT-3'；
5'- TGGTCTGACTGCTTGCTCTT -3'；
(17) the PCR reaction primers for detecting the CCNE1 gene have the following primer sequences:
5'- CTCAACGTGCAAGCCTCG -3'；
5'- CTCAAGAAAGTGCTGATCCC -3'；
(18) the PCR reaction primers for detecting the CCNG1 gene have the following primer sequences:
5'- AGTCTGCACACGATAATGGC -3'；
5'- TGCTTGGGCTGTACCTTCA -3'；
(19) the PCR reaction primer for detecting the CCNH gene has the following primer sequence:
5'- GGCACTTGAACAGATACTGGA -3'；
5'- CAATATGGGATAGCGGGTCT -3'；
(20) PCR primers for detecting CDC25A gene have the following primer sequences:
5'- TCCTCTTTTTACACCCCAGTCA -3'；
5'- CGGTTGTCAAGGTTTGTAGTTC -3'；
(21) PCR primers for detecting CDC25C gene have the following primer sequences:
5'- TGACAATGGAAACTTGGTGGAC-3'；
5'-GAGCGATATAGGCCACTTCTG -3'；
(22) PCR primers for detecting CDK1 gene have the following primer sequences:
5'-GATGTGCTTATGCAGGATTCC -3'；
5'- ATGTACTGACCAGGAGGGATAG -3'；
(23) PCR primers for detecting CDK4 gene have the following primer sequences:
5'- TGGTGTTTGAGCATGTAGACC -3'；
5'- ATCCTTGATCGTTTCGGCTG -3'；
(24) the PCR reaction primers for detecting the CDKN1A gene have the following primer sequences:
5'- GATGGAACTTCGACTTTGTCA -3'；
5'- CACAAGGGTACAAGACAGTG -3'；
(25) the PCR reaction primers for detecting the CDKN2A gene have the following primer sequences:
5'- TGGAGCCTTCGGCTGACT -3'；
5'- TAACTATTCGGTGCGTTGGG -3'；
(26) the PCR reaction primers for detecting the CHEK1 gene have the following primer sequences:
5'- CTTACTGCAATGCTCGCTGG -3'；
5'-TGAGGGGTTTGTTGTACCATC-3'；
(27) the PCR reaction primers for detecting the CHEK2 gene have the following primer sequences:
5'- TATCTGCCTTAGTGGGTATCCA -3'；
5'- TGTCGTAAAACGTGCCTTTG -3'；
(28) the PCR reaction primer for detecting CRDD gene has the following primer sequence:
5'- ATCAGACCGGCAGATTAACC -3'；
5'- TTGGCCTTACAGCGGTAGAT -3'；
(29) the PCR reaction primers for detecting the DNMT1 gene have the following primer sequences:
5'- CTAGCCCCAGGATTACAAGG -3'；
5'- CTCATCCGATTTGGCTCTTTC -3'；
(30) the PCR reaction primers for detecting the E2F1 gene have the following primer sequences:
5'- ATCCCAGGAGGTCACTTCTG-3'；
5'- ACAACAGCGGTTCTTGCTC-3'；
(31) the PCR reaction primers for detecting the E2F3 gene have the following primer sequences:
5'- AAGCCCCTCCAGAAACAAGA -3'；
5'- CTTGGGTACTTGCCAAATGT -3'；
(32) the PCR reaction primers for detecting the EGFR gene have the following primer sequences:
5'- TGCCGCAAAGTGTGTAACG -3'；
5'- TGCCGCAAAGTGTGTAACG -3'；
(33) the PCR reaction primers for detecting the EGR1 gene have the following primer sequences:
5'-CCCCTCTGTCTACTATTAAGGC -3'；
5'- GGGACTGGTAGCTGGTATTG-3'；
(34) the PCR primers for detecting the EI24 gene have the following primer sequences:
5'- TCGGTAACAGCCCGAATTATC -3'；
5'- CACTGAAAATTGACGTGAGGAA -3'；
(35) the PCR reaction primer for detecting the ESR1 gene has the following primer sequence:
5'- AAAGGTGGGATACGAAAAGACC-3'；
5'- CTGTTCTTCTTAGAGCGTTTGA -3'；
(36) the primer sequence of the PCR primer for detecting the FADD gene is as follows:
5'- TGGCTGACCTGGTACAAGAG -3'；
5'- GTAGATGCGTCTGAGTTCCAT -3'；
(37) the primer sequence of the PCR reaction primer for detecting the FAS gene is as follows:
5'- GATTGTGTGATGAAGGACATGG -3'；
5'- GTTGCTGGTGAGTGTGCATT -3'；
(38) the primer sequence of the PCR reaction primer for detecting the FASLG gene is as follows:
5'- TTTAACAGGCAAGTCCAACTCA -3'；
5'- GCCACCCTTCTTATACTTCACT -3'；
(39) the primer sequence of the PCR reaction primer for detecting the FOXO3 gene is as follows:
5'- CGGCTGACTGATATGGCAG -3'；
5'- GTGATGTTATCCAGCAGGTC -3'；
(40) the PCR reaction primers for detecting the GADD45A gene have the following primer sequences:
5'- CCTGATCCAGGCGTTTTG-3'；
5'- ATCCATGTAGCGACTTTCCC-3'；
(41) the PCR reaction primer for detecting the GML gene has the following primer sequence:
5'- TCTTTGCCTTACTCCTAGCCA -3'；
5'- GCGCCTAATATGATACGGACA -3'；
(42) the primer sequence of the PCR reaction primer for detecting the HDAC1 gene is as follows:
5'-GCCCTCACAAAGCCAATG -3'；
5'- TGCTTGCTGTACTCCGACA -3'；
(43) the PCR reaction primers for detecting the HK2 gene have the following primer sequences:
5'- TGACCAGGAGATTGACATGGG -3'；
5'- AACCGCATCAGGACCTCA -3'；
(44) the PCR reaction primer for detecting IGF1R gene has the following primer sequence:
5'- TGCTGACCTCTGTTACCTCT-3'；
5'- GCTTATTCCCCACAATGTAGTT-3'；
(45) the primer sequence of the PCR reaction primer for detecting the IL6 gene is as follows:
5'- CTGAACCTTCCAAAGATGGC-3'；
5'- TCACCAGGCAAGTCTCCTCA-3'；
(46) the PCR reaction primer for detecting the JUN gene has the following primer sequence:
5'- ACAGGTGGCACAGCTTAAAC-3'；
5'- AACTGCTGCGTTAGCATGAG-3'；
(47) the PCR reaction primers for detecting the KAT2B gene have the following primer sequences:
5'- GGAAAACCTGTGGTTGAAGG -3'；
5'- AGTCTTCGTTGAGATGGTGC-3'；
(48) the primer sequence of the PCR reaction primer for detecting the KRAS gene is as follows:
5'- AGTACAGTGCAATGAGGGAC -3'；
5'- CTGAGCCTGTTTTGTGTCTAC -3'；
(49) the PCR reaction primers for detecting the MCL1 gene have the following primer sequences:
5'- TGCCTTTGTGGCTAAACACT-3'；
5'- GTCCCGTTTTGTCCTTACGA-3'；
(50) PCR primers for detecting MDM2 gene, the primer sequence is as follows:
5'- GCAGGGGAGAGTGATACAGA-3'；
5'- AAGCCAATTCTCACGAAGGG -3'；
(51) PCR primers for detecting MDM4 gene, the primer sequence is as follows:
5'- TTTGATCCCTGCAACTCAGTG -3'；
5'- TCTCGTGGTCTTTTCTCACAT -3'；
(52) the PCR reaction primers for detecting MLH1 gene have the following primer sequences:
5'- CAAACCCCTGTCCAGTCAG -3'；
5'- TGGGAGTTCAAGCATCTCCT -3'；
(53) the PCR reaction primers for detecting the MSH2 gene have the following primer sequences:
5'- GTCAGAGCCCTTAACCTTTTTC -3'；
5'- AGAGGCTGCTTAATCCACTG -3'；
(54) the PCR reaction primers for detecting MYC genes have the following primer sequences:
5'- TCAAGAGGCGAACACACAAC -3'；
5'- TGGACGGACAGGATGTATGC -3'；
(55) PCR primers for detecting MYOD1 gene have the following primer sequences:
5'- GGACGTGCCTTCTGAGTC -3'；
5'- GCACCTGGTATATCGGGTTG -3'；
(56) the primer sequence of the PCR reaction primer for detecting the NF1 gene is as follows:
5'- GAATCATCACCAATTCCGCA -3'；
5'- CACAACCTTGCACTGCTTTAT -3'；
(57) PCR reaction primers for detecting NFKB1 gene have the following primer sequences:
5'- GTGCGGCTCATGTTTACAG -3'；
5'- ATGGCGTCTGATACCACGG -3'；
(58) the PCR reaction primer for detecting the PCNA gene has the following primer sequence:
5'- CACTAAGGGCCGAAGATAACG -3'；
5'- CAGCATCTCCAATATGGCTGA -3'；
(59) the PCR reaction primer for detecting the PIDD gene has the following primer sequence:
5'- TCACCCACCTGTACGCAC-3'；
5'- AGAGCGATGAGGTTCACAC-3'；
(60) the PCR reaction primers for detecting the PPM1D gene have the following primer sequences:
5'- TGGTCATCATTCGGGGCAT -3'；
5'- ATCCTTCGGGTCATCCTGAA -3'；
(61) the PCR reaction primers for detecting the PRC1 gene have the following primer sequences:
5'- AGTGGAATTGATGCGAAAACAG -3'；
5'- CTCACGCCTAGAAGCCTTTG -3'；
(62) the PCR reaction primer for detecting PRKCA gene has the following primer sequence:
5'-TGTCACAGTACGAGATGCAAAA -3'；
5'- CTTTCATTCTTGGGATCAGGAA -3'；
(63) the primer sequence of the PCR reaction primer for detecting the PTEN gene is as follows:
5'- GGGACGAACTGGTGTAATGA -3'；
5'- TGGTCCTTACTTCCCCATAGAA -3'；
(64) the PCR reaction primers for detecting the PTTG1 gene have the following primer sequences:
5'- CCCGTGTGGTTGCTAAGG -3'；
5'- CGTGGTGTTGAAACTTGAGAT-3'；
(65) the PCR reaction primers for detecting the RB1 gene have the following primer sequences:
5'- ACTTTGTGAACGCCTTCTGT -3'；
5'- ACGTTTGAATGTCTCCTGAACA -3'；
(66) the primer sequence of the PCR reaction primer for detecting the RELA gene is as follows:
5'- TGGGGACTACGACCTGAATG-3'；
5'- GGGCACGATTGTCAAAGATG -3'；
(67) the PCR reaction primers for detecting the SESN2 gene have the following primer sequences:
5'- CTTACCTGGTAGGCTCCCAC -3'；
5'- GCAACTTGTTGATCTCGCTG -3'；
(68) the PCR reaction primers for detecting the SIAH1 gene have the following primer sequences:
5'- CCATTCGCAACTTGGCTATG -3'；
5'- GGCACGGACAGGAATAAGG -3'；
(69) the PCR reaction primers for detecting the SIRT1 gene have the following primer sequences:
5'- GTGTCATAGGTTAGGTGGTGA -3'；
5'- GCCAATTCTTTTTGTGTTCGTG -3'；
(70) the PCR reaction primer for detecting the STAT1 gene has the following primer sequence:
5'- GGCTGAATTTCGGCACCT -3'；
5'- GGCTGAATTTCGGCACCT-3'；
(71) the PCR reaction primers for detecting the TADA3 gene have the following primer sequences:
5'- CTGATGACCCTATCGACGTG -3'；
5'- CAGGGGTGGGATCTTGTAAT -3'；
(72) the PCR reaction primer for detecting TNF gene has the following primer sequence:
5'- AGGCCAAGCCCTGGTATG-3'；
5'- GGGCCGATTGATCTCAGC -3'；
(73) the PCR reaction primers for detecting the TNFRSF10B gene have the following primer sequences:
5'- CAGTTGCAGCCGTAGTCTTG -3'；
5'- CAGGTCGTTGTGAGCTTCT-3'；
(74) the PCR reaction primers for detecting the TNFRSF10D gene have the following primer sequences:
5'- TTGGCTTTTCATGTCGGAAGA -3'；
5'- CCAGGAACTCGTGAAGGAC -3'；
(75) the primer sequence of the PCR reaction primer for detecting the TP53 gene is as follows:
5'- CAGCTTTGAGGTGCGTGTTT -3'；
5'- CCTTTCTTGCGGAGATTCTCT -3'；
(76) the primer sequence of the PCR reaction primer for detecting the TP53AIP1 gene is as follows:
5'- CCTGGCTGGGTAAGTCCC -3'；
5'- TCCTGGTGAGTCTGAAAACTTG -3'；
(77) the primer sequence of the PCR reaction primer for detecting the TP53BP2 gene is as follows:
5'- AAGTGCGTCCGTTCTCAATG -3'；
5'- GTTCTTCCTCAGAGTACCAAAG -3'；
(78) the primer sequence of the PCR reaction primer for detecting the TP63 gene is as follows:
5'- TCATTTGATTCGAGTAGAGGGG -3'；
5'- TGGGGTGGCTCATAAGGT -3'；
(79) the primer sequence of the PCR reaction primer for detecting the TP73 gene is as follows:
5'- ACGAGGACACGTACTACCTT-3'；
5'- TGCCGATAGGAGTCCACCA -3'；
(80) the primer sequence of the PCR reaction primer for detecting the TRAF2 gene is as follows:
5'- CTCATGCTGACCGAATGTC-3'；
5'- CCGTCACAAGTTAAGGGGAA -3'；
(81) the PCR reaction primers for detecting the TSC1 gene have the following primer sequences:
5'- TGGCGGAAGTCTATCTCGTC-3'；
5'- CAAGGGTACATTCCATAAAGGC-3'；
(82) the primer sequence of the PCR reaction primer for detecting the WT1 gene is as follows:
5'- TGACTTCAAGGACTGTGAACG -3'；
5'- GGGAGAACTTTCGCTGACAA -3'；
(83) the PCR reaction primers for detecting the XRCC5 gene have the following primer sequences:
5'- GACTTCCTGGATGCACTAATCG -3'；
5'- CTAAGCGAAAGGGGCCAT -3'；
(84) the PCR reaction primer for detecting the RPRM gene has the following primer sequence:
5'-TGAACGCGACCTTCCTGAA-3'；
5'- ACCACGGTAAGCGACAG -3'；
(85) the PCR reaction primers for detecting the ACTB gene have the following primer sequences:
5'- ATGTACGTTGCTATCCAGGC-3'；
5'- TCCTTAATGTCACGCACGAT-3'；
(86) the PCR reaction primers for detecting the B2M gene have the following primer sequences:
5'- AGGCTATCCAGCGTACTCCA-3'；
5'-GGCAGGCATACTCATCTTTT -3'；
(87) the PCR reaction primers for detecting the GAPDH gene have the following primer sequences:
5'- TGGGCTACACTGAGCACC-3'；
5'- AGTGGTCGTTGAGGGCAATG-3'；
(88) the PCR reaction primers for detecting the HPRT1 gene have the following primer sequences:
5'-AAAAGGACCCCACGAAGTGT -3'；
5'- GTCAAGGGCATATCCTACAACA-3'；
(89) the PCR reaction primers for detecting the RPLP0 gene have the following primer sequences:
5'- AGATTGGCTACCCAACTGTT-3'；
5'- GAAGGTGTAATCCGTCTCCAC-3'。
2. use of the PCR reagent of claim 1 in the preparation of a kit for detecting the core molecule of the P53 signaling pathway of cell line.
3. The use of the PCR reagent according to claim 2 in the preparation of a kit for detecting the core molecule of the P53 signaling pathway of cell line, characterized in that: the cell lines are human oral squamous carcinoma cells, normal oral epithelial cells, liver cancer cells, gastric cancer cells, lung cancer cells, breast cancer cells and melanoma cells.
4. The use of the PCR reagent according to claim 2 in the preparation of a kit for detecting the core molecule of the P53 signaling pathway of cell line, characterized in that: the kit adopts a real-time fluorescent quantitative PCR method for detection.
5. The use of the PCR reagent according to claim 2 in the preparation of a kit for detecting the core molecule of the P53 signaling pathway of cell line, characterized in that: the reaction system of the real-time fluorescent quantitative PCR is as follows: 10 mul of Taq polymerase reaction mixture, 0.1 mul-0.2 mul of upstream primer, 0.1 mul-0.2 mul of downstream primer, 25ng-100ng of cDNA template, and sterile distilled water to make up to 20 mul.
6. The use of the PCR reagent according to claim 2 in the preparation of a kit for detecting the core molecule of the P53 signaling pathway of cell line, characterized in that: the Taq polymerase is 2X SYBR Premix Ex Taq polymerase of TaKaRa company.
7. The use of the PCR reagent according to claim 2 in the preparation of a kit for detecting the core molecule of the P53 signaling pathway of cell line, characterized in that: the reaction procedure of the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 deg.C for 5 seconds, 59 deg.C for 20 seconds, and 40 cycles; the dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.
CN202010666777.7A 2020-07-13 2020-07-13 PCR reagent for detecting molecular expression of cell P53 signal path and application thereof Pending CN111690750A (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN113278704A (en) * 2021-07-22 2021-08-20 北京泱深生物信息技术有限公司 Marker and product for diagnosing oral squamous cell carcinoma

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CN102251050A (en) * 2011-08-10 2011-11-23 青岛大学医学院附属医院 Reagent for detecting cell apoptosis signal path and PCR method using same

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CN102251050A (en) * 2011-08-10 2011-11-23 青岛大学医学院附属医院 Reagent for detecting cell apoptosis signal path and PCR method using same

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BRADLEY G等: "The expression of p53-induced protein with death domain (Pidd) and apoptosis in oral squamous cell carcinoma", 《BRITISH JOURNAL OF CANCER》 *
CAT.NO.330231 PAHS-027ZA: "RT2 Profiler PCR Array (96-Well Format and 384-Well [4×96] Format) Human p53 Signaling Pathway", 《QIAGEN》 *
WANG X等: "Downregulation of GBAS regulates oral squamous cell carcinoma proliferation and apoptosis via the p53 signaling pathway", 《ONCOTARGETS AND THERAPY》 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113278704A (en) * 2021-07-22 2021-08-20 北京泱深生物信息技术有限公司 Marker and product for diagnosing oral squamous cell carcinoma

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